The 26S proteasome is a large multisubunit complex comprised of a central core particle, which degrades any protein that can access its buried proteolytic sites, and flanking regulatory particles, which recognize and process polyubiquitylated proteins for translocation into the core particle for subsequent degradation. The 26S proteasome is an established target for cancer therapeutics, with inhibitors of the core particle proteolytic sites approved for the treatment of multiple myeloma and mantle cell lymphoma. Efficacy of these drugs is limited, however, by their toxicity and range of cancers that can be treated, primarily because proteolysis by the 26S proteasome is essential in eukaryotic cells. Recently, the proteasome regulatory particle subunit RPN13 has been reported to be a promising new cancer target in studies with cell culture and animal models using RA190, an electrophilic small molecule that covalently binds to RPN13. An appeal of targeting RPN13 is that, unlike the majority of proteasome subunits, it is not essential in yeast or mice, where deletion elicits only a mild phenotype, but does seem to be important for at least several types of cancer. Based upon our unpublished biochemical and structural data, we propose an alternative approach to inhibiting RPN13 that will employ a mechanism orthogonal to that of RA190 and seems likely to generate fewer side effects because: (1) it will not directly inhibit the essential catalytic sites in the core particle, (2) will not use a chemically reactive group, and () will not inhibit non-proteasomal roles of RPN13 (which also functions with the INO80 chromatin remodeler). Our foundational studies include determining a 1.45 A resolution crystal structure of a complex between human RPN13 and the RPN2 C-terminal residues, which our preliminary data show serve as the sole determinant of RPN13 association with the proteasome. We have also developed an SPR assay, experimentally determined the binding affinity between RPN13 and the RPN2 C-terminus to be 10nM, and mapped the primary binding determinant to two adjacent aromatic residues of RPN2 that sit in a deep pocket on RPN13 that includes only 240 A2 of contact surface area. Adding further confidence to our approach, we have shown that deletion of the binding peptide from RPN2 can displace RPN13 from purified proteasomes. Building upon these insights, this proposal seeks to establish inhibition of RPN13-proteasome association as an attractive target for novel cancer therapeutics. Toward this goal we are developing approaches to identify small molecule inhibitors of RPN13- proteasome association (Aim 1) and developing cell culture approaches to establish the consequences of disrupting RPN13-proteasome association (Aim 2) in several different cancer cell lines.